Title: Peritumoral Vascular Invasion and NHERF1 expression define an immunophenotype of grade 2 invasive breast cancer associated with poor prognosis

Transcription

1 Author's response to reviews Title: Peritumoral Vascular Invasion and NHERF1 expression define an immunophenotype of grade 2 invasive breast cancer associated with poor prognosis Authors: Andrea Malfettone (a.malfettone@gmail.com) Concetta Saponaro (titasap@hotmail.com) Angelo Paradiso (a.paradiso@oncologico.bari.it) Giovanni Simone (g.simone@oncologico.bari.it) Annita Mangia (a.mangia@oncologico.bari.it) Version: 2 Date: 30 December 2011 Author's response to reviews: see over

2 Please note that: All changes to our manuscript have been marked in yellow. Reviewer: Anna Sapino Reviewer 1 Reply Thus the words molecular subtype in the title should be changed in immunophenotype. We would like to thank the Referee for the meaningful comments and for the several suggestions, which arisen from a deep and critical revision of our manuscript. Regarding the first comment, in accord with the Referee s comment to avoid further misunderstandings, in the Title the definition molecular subtype has been replaced with immunophenotype, as follow: << Peritumoral Vascular Invasion and NHERF1 expression define an immunophenotype of grade 2 invasive breast cancer associated with poor prognosis >> Why the histological grade was available for 175 patients only? Of the 187 breast cancers included in this study, 12 cases were classic variant of Invasive Lobular Carcinoma, which could not be graded. Application of the grading system to this histological tumor type was not routine in histopathology reporting at time of diagnosis by pathologists of our Institute. The number of cases with a family history is quite high in this series, this means that a case selection was performed and this should be specified. Our selected series of 187 primary invasive breast cancers included in this study was composed by 97 familial and 90 sporadic cases. Following a specific genetic counseling program, patients were classified as having a family history of breast cancer utilizing criteria described previously (Bruno M, et al. Ann Oncol 2004;15:I Tommasi S, et al. Mutat Res 2005;578: Mangia A, et al. Histol Histopathol 2009;24:69-76). Moreover, DNA of each affected proband is currently being screened for mutations in the entire coding region of the BRCA1/2 genes. In future studies we will try to verify the influence of such hypothetical mutations, such as BRCA gene mutations, on prognosis of the G2 familial breast cancer subgroup. Therefore, in accordance with the Referee s comment, in the Patients and tumor specimens paragraph of the Method section, we have been specified the selection criteria of breast cancer patient cohort: << A selected series of 187 primary invasive breast carcinomas were included in this study: 48% (n = 90) were sporadic patients and 52% (n = 97) were classified as having a family history, after a genetic counseling program as reported previously [25]. None of the sporadic patients had a family history of breast or ovarian cancer. >> 1

3 The sentence Then, we applied the NPI to a series of well defined biological factors in order to establish the prognostic influence of each of these variables (page 5) is not clear. According to the Referee s suggestion, in the Patients and tumor specimens paragraph of the Method section we have modified the reported sentence with the following one: << Then, we verified whether breast cancers with Good, Moderate or Poor prognosis were associated with a series of well-defined biological factors and with tumor markers not currently used in routine diagnosis. >> The cut off of 40% to define positive cases is for both cytoplasmic and membrane expression? In the Immunohistochemistry paragraph of the Method section, we have integrated the mentioned sentence by the following: << According to median value cut-off, cases were classified positive when cytoplasmic NHERF1 immunoreactivity was present in >40% of tumor cells and when membranous NHERF1 expression was detected in >0% of tumor cells examined. >> The cut off of 20% for Mib1 should be justified. Since the lack of a worldwide agreed Mib1 cut off, the median value of MIB1 in breast carcinoma was reported to be less than 10%, between 10-20% or higher than 20%, according to several reports. It is thus recommended that each laboratory evaluates, with its internal procedures, the different aspects of the methodology to be used for MIB1 scoring (Fitzgibbons PL et al: Arch Pathol Lab Med 124(7): , 2000). In the present work, the MIB1 cut-off of 20% corresponds to the median value of the scores obtained by the analysis of all breast tumor samples during the last five years within our Institute. Therefore, this aspect has been explained in the Patients and tumor specimens paragraph of the Method section. Results related to G1 tumours should be given. Some biases of classification of G2 tumours may have been done, because of the clear cut inverse correlation with tumour size, nodal status and MIB1 and the direct correlation with ER and PR of G2 cancers. A revision of the series of G2 tumours will probably move at least some of them, to G1. After cases selection to be included in the study and before beginning analyses, all histological sections of tumor specimens were re-evaluated by two experienced pathologists, without any knowledge of patient history. Moreover, even a comprehensive revision of each tumor scored as G2 was made. As described in our manuscript, patients who have intermediate-grade tumors are the major source of inter-observer discrepancy and may display intermediate phenotype and survival, making treatment decisions a great challenge, with subsequent under- or over-treatment (M. Ignatiadis & C. Sotiriou, Pathobiology 2008;75: Rakha EA et al, J Clin Oncol. 2008;26(19): Rakha et al, Breast Cancer Research 2010, 12:207). In our series, we found a proportion of cases in each grade similar to that reported in different studies, where grade 1 tumors generally represent 11% to 38% of cases; grade 2, 30% to 60%; and grade 3, 19 to 46% 2

4 (Elston CW, Aust N Z J Surg 1984, 54: Davis BW et al, Cancer 1986, 58: Hopton DS et al, Eur J Surg Oncol 1989, 15: Le Doussal V et al, Cancer 1989, 64: Balslev I et al, Breast Cancer Res Treat 1994, 32: Saimura M et al, J Surg Oncol 1999, 71: Reed W et al, Cancer 2000, 88: Simpson JF et al, J Clin Oncol 2000, 18: Lundin J et al, J Clin Oncol 2001, 19: Frkovic-Grazio S & Bracko M, J Clin Pathol 2002, 55: Warwick J et al, Cancer 2004, 100: Williams C et al, Health Technol Assess 2006, 10:iii-iv, ix-xi, Rakha EA et al, J Clin Oncol 2008, 26: Thomas JS et al, Histopathology 2009, 55: Blamey RW et al, Eur J Cancer 2010, 46:56-71). In particular, of 187 invasive breast cancers included in our study, 46% (n=81) of tumors were histological grade 2, a percentage in the middle of the observed frequency. Grade 2 tumors usually show the lowest degree of concordance and this is an expected phenomenon of scoring of a biological variable where scores in the overlap regions are usually most difficult to be categorized. Some more information regarding the significance of cytoplasmic or membrane expression of NHERF1 should be given. Are they concomitant or one excludes the other? The Referee s comment is correct, in fact the different localization of NHERF1, in the plasma membrane and/or cytoplasm, has been better described in the Relationship between tumor markers and clinicopathological features paragraph of the Results section, as follows: << All breast cancers showed NHERF1 protein localized in the cytoplasm of tumor cells and 31% of overexpressing cytoplasmic NHERF1 tumors exhibited a significant association with tumor grade 3 (p = 0.029), negative PR status (p = 0.008), high MIB1 (p = 0.033), positive HER2 status (p = 0.025) and with moderate NPI (p = 0.038). In 13% of overexpressing membranous NHERF1 tumors, in addition to cytoplasmic immunoreactivity, NHERF1 showed also a plasma membrane localization; these overexpressing membranous NHERF1 tumors were significantly associated with tumor grade 2 (p = 0.014), positive PR status (p = 0.031), low MIB1 (p = 0.029) and with good NPI (p = 0.010). >> Moreover, in the Discussion section we have explained the biological implications of this different NHERF1 subcellular distribution, as follows: << The adaptor protein NHERF1 shows a physiological localization at the plasma membrane, but during breast cancerogenesis progressively loses its apical localization becoming mostly cytoplasmic in no longer polarized tumor cells [17]. In our series, 13% of tumors showed NHERF1 still localized in the plasma membrane, and were positively associated with favorable prognosis parameters, such as low tumor grade, positive PR status, and low proliferative activity. The positive prognostic impact of membranous NHERF1 is in agreement with results obtained from our [17,18] and other laboratories [46], suggesting that NHERF1 might behave either as a tumor suppressor, when it is localized at the plasma membrane, or as an oncogenic protein, when it is shifted to the cytoplasm, depending on its subcellular distribution. >> The results on the correlation with ER expression are not clear. The corresponding sentence in the Relationship between tumor markers and clinicopathological features paragraph of the Results section has been reformulated with the following one: 3

5 << Furthermore, correlation analysis further revealed that cytoplasmic NHERF1 expression levels were positively correlated with increasing ER levels (p = 0.022) (data not shown). >> The authors speak of tumour biomarker signature referring to the panel of antibodies used in the paper, but this is not correct. We have modified the aim paragraph in the Discussion section as follow: << In this study, we explored traditional prognostic factors and a panel of tumor markers not used in routine diagnosis, such as NHERF1, VEGFR1, HIF-1α and TWIST1, that have been respectively related to breast cancer progression [17-19], aggressiveness [21,22], hypoxic response [29,31] and cell invasion/metastasis [24,33], assessing if they are differentially expressed in tumors scored as grade 2, in order to best characterize them. >> They refer to ER-dependent correlation in positive ER tumours this sentence is not clear. The above-mentioned sentence has been modified as follow: << However, in line with several reports describing the NHERF1 gene as transcriptionally regulated by estrogen [18,36,37], here we demonstrated that cytoplasmic NHERF1 expression was significantly correlated with increasing levels of ER status. >> The first sentence of the conclusions should be modulated. To avoid any misunderstanding, the first sentence of the Conclusions paragraph in the Discussion section has been amended as follows: << We showed in this study that cytoplasmic NHERF1 colocalizes with the oncogenic receptor VEGFR1and their significant correlation suggests new potential implications in breast tumor progression. >> Vascular invasion may be either of lymphatic or blood vessels or of both so the presence of erythrocytes it is not a requisite for the diagnosis of vascular invasion. Our study has further confirmed the prognostic importance and validity of the peritumoral vascular invasion in breast cancer, a pathological characteristic recognized by the presence of tumor cells in vascular spaces closely associated with primary invasive mammary carcinoma. In agreement with Referee s advice, we have amended as follows the PVI sentence in the Patients and tumor specimens paragraph of the Materials and methods section: << Assessment of the peritumoral vascular invasion (PVI) was based upon examination of sections stained with haematoxylin and eosin and was considered evident if at least one cohesive clump of tumor cells was clearly visible within peritumoral endothelial-lined spaces, both lymphatic channels and small blood vessels - closely associated with primary invasive carcinoma [27]. >> Regarding HIF1 the authors should specified that the expression pattern (perinecrotic or diffuse) is typical of HIF1. 4

6 In the Immunohistochemistry paragraph of the Methods section, we have amended description of the HIF-1α expression pattern, as follows: <<HIF-1α was considered overexpressed when >0% of nuclei were positive and the typical expression pattern (perinecrotic or diffuse) was noted [29].>> For the evaluation of the percentage of VEGFR1 expression the sentence with respect to the total analysed is redundant. Other sentences are not necessary such as who had no patient information, considering only the invasive epithelial component of tumour at page 7. As suggested by the Referee, in the Immunohistochemistry paragraph of the Method section, all the mentioned sentences together with other ones have been properly revised. Why the authors used different antibodies against NHERF1 for double staining? The origin of the antibodies (mouse/rabbit) should be given. The Referee s comment is correct and in the Immunohistochemistry and Immunohistofluorescence paragraphs of the Method section have been specified all technical characteristics of the antibodies, as reported in their respective datasheets. In particular, with regard to anti-nherf1 antibody used for the Immunofluorescence double staining assay, we utilized a mouse monoclonal antibody already standardized and validated in our previous works (Cardone RA, Mol Biol Cell 2007;18(5): Mangia A, Histopathology 2009;55(5): Bellizzi A, Histopathology 2011;58(7): ). Are the data on prognosis related to G2 cancers classified by Elston and Ellis grade or by NPI? As reported in the Patients and tumor specimens paragraph of the Method section, the histogical grading for all invasive tumors included in this study was performed according to the Elston and Ellis method. 5

7 Please note that: All changes to our manuscript have been marked in yellow. Reviewer Anne Vincent-Salomon Referee 2 Reply Although this aim is of key importance in clinical practice, the authors should focuse their analyses on a series of consecutive grade 2, with no enrichment in familial cases as in their chosen population of cases. We would like to thank the Referee for the meaningful comments and for the several suggestions, which arisen from a deep and critical revision of our manuscript. With regard to the first question, we would clarify that our selected series of 187 primary invasive breast cancers included in this study was composed by 97 familial and 90 sporadic cases. Following a specific genetic counseling program, patients were classified as having a family history of breast cancer utilizing criteria described previously (Bruno M, et al. Ann Oncol 2004;15:I Tommasi S, et al. Mutat Res 2005;578: Mangia A, et al. Histol Histopathol 2009;24:69-76). Moreover, DNA of each affected proband is currently being screened for mutations in the entire coding region of the BRCA1/2 genes. In future studies we will try to verify the influence of such hypothetical mutations, such as BRCA gene mutations, on prognosis of the G2 familial breast cancer subgroup. Therefore, in accordance with the Referee s comment, in the Patients and tumor specimens paragraph of the Method section, we have been specified the selection criteria of breast cancer patient cohort: << A selected series of 187 primary invasive breast carcinomas were included in this study: 48% (n = 90) were sporadic patients and 52% (n = 97) were classified as having a family history, after a genetic counseling program as reported previously [25]. None of the sporadic patients had a family history of breast or ovarian cancer. >> However, the proposed explanations for these markers (NHEF1, TWIST and VEGFR1) need to be more clearly stated. The link between NHEF1 and HIF1alpha is not obvious to the reviewer and should be clarified. Identically, links between TWIST and VEGFR1 and HIF1 are also too elusive. The authors should develop why these markers are the pertinent ones to analyze to decipher grade 2 tumors into groups of different outcome. The aim of the present paper was to distinguish good prognosis and poor prognosis cases in the vast subgroup of grade 2 breast carcinomas. In addition to the traditional prognostic parameters, we have considered several immunohistochemical markers that are not used in routine diagnosis, but that have been correlated with prognosis in previous studies. Therefore, we explored a panel of tumor markers associated with key biological processes in breast cancer: tumor progression (NHERF1), aggressiveness (VEGFR), hypoxic response (HIF-1α) and cell invasion/metastasis (TWIST). In this regard, on the basis of Referee's suggestions several corrections have been made in the manuscript. 6

8 The analyzed population, enriched with familial cases, is also enriched with N+ cases that doesn't reflect classically observed N+ rates for T2 cases (around 30 to 40%). After cases selection to be included in the study and before beginning analyses, all histological sections of tumor specimens were re-evaluated by two experienced pathologists, without any knowledge of patient history. Moreover, even a comprehensive revision of each tumor scored as G2 was made. As described in our manuscript, patients who have intermediate-grade tumors are the major source of inter-observer discrepancy and may display intermediate phenotype and survival, making treatment decisions a great challenge, with subsequent under- or over-treatment (M. Ignatiadis & C. Sotiriou, Pathobiology 2008;75: Rakha EA et al, J Clin Oncol. 2008;26(19): Rakha et al, Breast Cancer Research 2010, 12:207). In our series, we found a proportion of cases in each grade similar to that reported in different studies, where grade 1 tumors generally represent 11% to 38% of cases; grade 2, 30% to 60%; and grade 3, 19 to 46% (Elston CW, Aust N Z J Surg 1984, 54: Davis BW et al, Cancer 1986, 58: Hopton DS et al, Eur J Surg Oncol 1989, 15: Le Doussal V et al, Cancer 1989, 64: Balslev I et al, Breast Cancer Res Treat 1994, 32: Saimura M et al, J Surg Oncol 1999, 71: Reed W et al, Cancer 2000, 88: Simpson JF et al, J Clin Oncol 2000, 18: Lundin J et al, J Clin Oncol 2001, 19: Frkovic-Grazio S & Bracko M, J Clin Pathol 2002, 55: Warwick J et al, Cancer 2004, 100: Williams C et al, Health Technol Assess 2006, 10:iii-iv, ix-xi, Rakha EA et al, J Clin Oncol 2008, 26: Thomas JS et al, Histopathology 2009, 55: Blamey RW et al, Eur J Cancer 2010, 46:56-71). In particular, of 187 invasive breast cancers included in our study, 46% (n=81) of tumors were histological grade 2, a percentage in the middle of the observed frequency. Grade 2 tumors usually show the lowest degree of concordance and this is an expected phenomenon of scoring of a biological variable where scores in the overlap regions are usually most difficult to be categorized. Details regarding the other histological types would be interesting to give and moreover, the level of TWIST expression in lobular carcinomas. In a previous report, a statistically significant correlation between Twist expression and invasive lobular carcinoma was found [Yang J, Cell 117 (2004)]. In the present paper, we have tried to find a potential correlation between the TWIST1 protein expression levels and various histological types, including the lobular carcinomas. However, any significant difference was found, because of the small number of lobular carcinomas compared to other histological types, or because of different antibodies/methodologies utilized. In the discussion, the authors should focused on the initial aim (to improve grade 2 tumor prognosis definitions). Statements given regarding biological hypothesis between high NHERF1 and cytoplasmic VEGFR1 are too hazardous and should rely on in vitro experiments. They are not under the scope of this work. In accord with Referee s comment, we have modified the Discussion section. 7

9 Moreover, to avoid any misunderstanding, the first sentence of the Conclusions paragraph in the Discussion section has been amended as follows: << We showed in this study that cytoplasmic NHERF1 colocalizes with the oncogenic receptor VEGFR1and their significant correlation suggests new potential implications in breast tumor progression. >> What is the correlation between proliferation and membranous NHERF1 and PVI specifically in the grade 2 tumors? This should be the central core of the paper. During the first elaboration of the manuscript, we have focused our attention on the potential association between the PVI/membranous NHERF1 expression immunophenotypes and the proliferation. On the whole series of patients, we found that the highly proliferative tumors were significantly associated to PVI+/membranous NHERF1- subgroup with poor prognosis (87% vs 13%, PVI+/membranous NHERF1- vs OTHERS, respectively, p = ). Although, the same immunophenotype didn t show a statistical correlation with proliferation in grade 2 tumors. The HER2 2+ and 3+ positive cases rate is extremely high. And suggest putative technical errors. The 2+ cases should be given separately from the 3+ cases and separated into HER2 amplified and HER2 non amplified with a FISH assessment of HER2 copy numbers. The Refereer s comment is correct, but in our study HER2 status was assessed exclusively for research purposes and scores 2+ and 3+ were considered positive for HER2 overexpression, accordingly to other works (Yamamoto Y, Breast Cancer Res Treat 110: , 2008; Seidman, J Clin Oncol 19: , 2001). 8

10 Please note that: All changes to our manuscript have been marked in yellow. Reviewer: Caterina Marchiò Referee 3 Reply Was the cohort of 187 breast carcinomas a series of consecutive carcinomas or were they somehow selected? If so, what were the selection criteria? We would like to thank the Referee for the meaningful comments and for the several suggestions, which arisen from a deep and critical revision of our manuscript. With regard to the first question, we would clarify that our selected series of 187 primary invasive breast cancers included in this study was composed by 97 familial and 90 sporadic cases. Following a specific genetic counseling program, patients were classified as having a family history of breast cancer utilizing criteria described previously (Bruno M, et al. Ann Oncol 2004;15:I Tommasi S, et al. Mutat Res 2005;578: Mangia A, et al. Histol Histopathol 2009;24:69-76). Moreover, DNA of each affected proband is currently being screened for mutations in the entire coding region of the BRCA1/2 genes. In future studies we will try to verify the influence of such hypothetical mutations, such as BRCA gene mutations, on prognosis of the G2 familial breast cancer subgroup. Therefore, in accordance with the Referee s comment, in the Patients and tumor specimens paragraph of the Method section, we have been specified the selection criteria of breast cancer patient cohort: << A selected series of 187 primary invasive breast carcinomas were included in this study: 48% (n = 90) were sporadic patients and 52% (n = 97) were classified as having a family history, after a genetic counseling program as reported previously [25]. None of the sporadic patients had a family history of breast or ovarian cancer. >> In the abstract and in the background please add HER2 overexpression in the list of breast cancer prognostic factors. The Abstract and the Background section have been modified following the Referee s suggestion. In the paragraph Prognosis analysis I would suggest to rephrase the following sentence: We examined whether tumors with grade 2 were associated with some distinct expression profile, as the authors are not actually evaluating an expression profile, given that clinicopathological variables are included in the list following the sentence above. According to the Referee s suggestion, we have modified both the Patients and tumor specimens paragraph of the Method section and the Prognosis analysis paragraph of the Results section with the following sentences, respectively: << Then, we verified whether breast cancers with Good, Moderate or Poor prognosis were associated with a series of well-defined biological factors and with tumor markers not currently used in routine diagnosis. >> 9

11 << We examined if tumors with grade 2 and poor prognosis were associated with some distinct clinicopathological parameters or with some tumor markers not currently used in routine diagnosis. >> Moreover, the words expression profiles in the text have been changed with immunophenotype. I would suggest the authors to present all data regarding statistical correlations with PVI and NHERF1 expression, as they represent the main core of the paper (see data not shown in the Prognosis Analysis section). In accord with Referee s suggestion, we have added the Figure 2C concerning statistical associations between the PVI/membranous NHERF1 immunophenotypes and the NPI in the whole cohort of 187 patients, and the Figure 2D concerning associations between the PVI/membranous NHERF1 immunophenotypes in grade 2 tumors both of familial and of sporadic patients. In addition, it is not clear whether a multivariate analysis was performed also for grade 2 tumours, in which presence of vascular invasion and loss of membranous NHERF1 were significantly associated with poor prognosis. Multivariate analysis is only described in details at the end of the Prognosis Analysis section and refers to the whole cohort. This should be specified as it is quite confusing in the current description of results. As already reported in each table of our manuscript and in the Figure2 legend, all G2 tumors subgroup analysis was performed utilizing the Fisher s exact, the Pearson χ 2 or χ 2 test as appropriate. However, in addition to previous statistical tests, univariate and multivariate logistic regression analyses were performed to evaluate the associations between tumor markers and NPI only on 187 breast cancers. In the prognosis analysis the authors state: Subgroup analysis revealed that the PVI (p = 0.026) and negative membranous NHERF1 (p = 0.033) were adverse prognostic factors for grade 2 tumors. Please add expression to the locution negative membranous NHERF1, then specify whether in these cases without membranous NHERF1 another type of expression was present (cytoplasmic?). In the Prognosis analysis paragraph of the Results section and in other parts of the manuscript, we have added the word expression to all sentences regarding the NHERF1 protein, in accord with the Referee s suggestion. However, the other Referee s comment is correct, in fact the different localization of NHERF1, in the plasma membrane and/or cytoplasm, has been better described in the Relationship between tumor markers and clinicopathological features paragraph of the Results section, as follows: << All breast cancers showed NHERF1 protein localized in the cytoplasm of tumor cells and 31% of overexpressing cytoplasmic NHERF1 tumors exhibited a significant association with tumor grade 3 (p = 0.029), negative PR status (p = 0.008), high MIB1 (p = 0.033), positive HER2 status (p = 0.025) and with moderate NPI (p = 0.038). In 13% of overexpressing membranous NHERF1 tumors, in addition to cytoplasmic immunoreactivity, NHERF1 showed also a plasma membrane localization; these overexpressing membranous NHERF1 tumors were significantly associated with 10

12 tumor grade 2 (p = 0.014), positive PR status (p = 0.031), low MIB1 (p = 0.029) and with good NPI (p = 0.010). >> Moreover, in the Discussion section we have explained the biological implications of this different NHERF1 subcellular distribution, as follows: << The adaptor protein NHERF1 shows a physiological localization at the plasma membrane, but during breast cancerogenesis progressively loses its apical localization becoming mostly cytoplasmic in no longer polarized tumor cells [17]. In our series, 13% of tumors showed NHERF1 still localized in the plasma membrane, and were positively associated with favorable prognosis parameters, such as low tumor grade, positive PR status, and low proliferative activity. The positive prognostic impact of membranous NHERF1 is in agreement with results obtained from our [17,18] and other laboratories [46], suggesting that NHERF1 might behave either as a tumor suppressor, when it is localized at the plasma membrane, or as an oncogenic protein, when it is shifted to the cytoplasm, depending on its subcellular distribution. >> As described by the authors from the very beginning of the paper the grade 2 subgroup of tumours included in this study is enriched for familial carcinomas. The authors should specify whether this means a positive genetic pedigree for the patients (i.e.: hereditary breast cancer) or a known breast disease in the family known by clinical query only. With regard to this aspect, please refer to our reply of the first comment. In addition, was a subgroup analysis of only grade 2 familial carcinomas performed? This becomes apparent from the discussion but is not well specified in the method and result section. Please amend this accordingly. In accord with Referee s suggestion, we have added and commented the Figure 2D concerning associations between the PVI/membranous NHERF1 immunophenotypes in grade 2 tumors of both familial and sporadic breast cancers. Were correlations between PVI and histological special types performed? Yes, we did. However, any statistical association was found. It is not clear whether estrogen and progesterone receptors were evaluated by immunoistochemistry in the present study or whether data on their expression in this cohort were taken from the pathological diagnostic reports, please specify. For all clinicopathological features of breast cancer patients, we have referred to data on the histological diagnosis and pathological reports from the Pathology Department of our Institute. In accord with Referee s suggestion, this aspect has been reported in the Patients and tumor specimens paragraph of the Methods section, as follow: 11

13 << Information regarding patient characteristics, including age, tumor size, nodal status, tumor grade, histologic tumor type, PVI, MIB1, ER and PR status, was collected from the Pathology Department of our Institute. >> I would avoid to use the term signature in a IHC study of a bunch of proteins (see discussion section, para 2: In this study, we explored traditional prognostic factors and tumor biomarker signature, ); please rephrase the sentence. The aim of the present paper was to distinguish good prognosis and poor prognosis cases in the vast subgroup of grade 2 breast carcinomas. In addition to the traditional prognostic parameters, we have considered several immunohistochemical markers that are not used in routine diagnosis, but that have been correlated with prognosis in previous studies. Therefore, we explored a panel of tumor markers associated with key biological processes in breast cancer: tumor progression (NHERF1), aggressiveness (VEGFR), hypoxic response (HIF-1α) and cell invasion/metastasis (TWIST). In this regard, on the basis of Referee's suggestions several corrections have been made in the Discussion section as follow: << In this study, we explored traditional prognostic factors and a panel of tumor markers not used in routine diagnosis, such as NHERF1, VEGFR1, HIF-1α and TWIST1, that have been respectively related to breast cancer progression [17-19], aggressiveness [21,22], hypoxic response [29,31] and cell invasion/metastasis [24,33], assessing if they are differentially expressed in tumors scored as grade 2, in order to best characterize them. >> Was the expression of HIF1-alfa found only along the membrane and in the cytoplasm? No nuclear expression detected? With regard to HIF1-alfa protein expression, only cells both with completely and darkly stained epithelial nuclei and with the typical expression pattern (perinecrotic or diffuse) were regarded as positive. Cytoplasmic staining, observed occasionally, was ignored for the immunohistochemical analysis. However, any membranous localization of HIF1-alfa protein was detected. Minor Essential Revisions Finally, we have completely revised the manuscript, giving particular attention to several typos and language mistakes throughout the paper. 12